This invention relates to a newly identified Src oncogene mutation, polypeptides containing such mutation, and polynucleotides encoding such mutation. This invention also relates to methods of identifying the Src mutation, to the use of such methods in therapy and diagnosis, and to methods of identifying agonist and antagonist compounds useful for treating and/or preventing clinical conditions associated with or caused by Src mutation. Methods and compositions are provided for identifying and treating malignant cells in a host such as human. Mutated DNA sequence probes and primers are made for determining the expression of mutated nucleic acid.
The discovery of Rous sarcoma virus (RSV) led to the identification of a cellular oncogene Src (c-Src) (SEQ ID NO. 1), which encodes a non-receptor tyrosine kinase (phosphoprotein of molecular weight 60,000 Dalton or pp60c-Src) (SEQ ID NO. 2). The Src oncogene has been implicated in the development of numerous types of cancers via a yet to be elucidated mechanism (see for example Stehelin, D., Varmus, H. E., Bishop, J. M. and Vogt, P. K. Nature 260, 170-173 (1976); Brugge, J. S. and Erikson, R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature 269, 346-348 (1977); Jove, R. and Hanafusa, H. Cell transformation by the viral Src oncogene. Annu Rev Cell Biol 3, 31-56 (1987); Thomas, S. M. and Brugge, J. S. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13, 513-609 (1997)). The nucleic acid sequence of normal c-Src is as follows:
The c-Src nucleic acid sequence (SEQ ID NO. 1) encodes for a tyrosine kinase protein pp60, which has a following sequence:
Amino acids are abbreviated as 1-letter codes and corresponding 3-letter codes as follows: Alanine is A or Ala; Arginine R or Arg, Asparagine N or Asn; Aspartic acid D or Asp; Cysteine C or Cys; Glutamine Q or Gln; Glutamic acid E or Glu; Glycine G or Gly; Histidine H or His; Isoleucine I or Ile; Leucine L or Leu; Lysine K or Lys; Methionine M or Met; Phenylalanine F or Phe; Proline P or Pro; Serine S or Ser; Threonine T or Thr; Tryptophan W or Trp; Tyrosine Y or Tyr; and Valine V or Val.
The cellular Src oncogene (c-Src) (SEQ ID NO. 1) is the normal counterpart of the transforming viral Rous sarcoma oncogene (v-Src). v-Src has been shown to induce the production of specific metalloproteinases (Hamaguchi, M. et al. Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of Src. Oncogene 10, 1037-1043 (1995)) and to foster the metastatic phenotype (Egan, S. et al. Transformation by oncogenes encoding protein kinases induces the metastatic phenotype. Science 238 202-205 (1987); Tatsuka, M. et al. Different metastatic potentials of ras- and Src-transformed BALB/c 3T3 A31 variant cells. Mol. Carcinog. 15, 300-308 (1996)). However, as opposed to cellular c-Src (SEQ ID NO. 1) the retroviral v-Src has 19 C-terminal residues replaced by a sequence of 12 amino acids, lacking the regulatory tyrosine.
The non receptor tyrosine kinase c-Src consists of an SH3, SH2 and tyrosine kinase domain. c-Src appears to be the most important to the normal function of osteoclasts, as determined from studies of Src-knock-out mice (see for example U.S. Pat. No. 5,541,109). The catalytic activity of c-Src and other nonreceptor tyrosine kinases is inhibited by the intramolecular association of their intrinsic SH2 domain to the carboxy-terminal tail upon phosphorylation of Tyr (position 530, avian position 527). Protein tyrosine phosphorylation is believed to be an important regulatory event in cell growth and differentiation. Phosphorylation on tyrosine can either decrease or increase the enzymatic activity of substrate proteins. Tyrosine phosphorylated sequences associate with Src homology 2 (SH2) domains, and thus tyrosine phosphorylation also serves to regulate protein/protein interactions. Many protein tyrosine kinases have been described to date: several are the receptors for peptide growth factors; others are expressed in the cytoplasm and nucleus. Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). There are 19 known families of receptor tyrosine kinases including the Her family (EGFR, Her 2, Her 3, Her 4), the insulin receptor family (insulin receptor, IGF-1R, insulin-related receptor), the PDGF receptor family (PDGF-R alpha and beta, CSF-1R, Kit, Flk2), the Flk family (Flk-1, Flt-1, Flk-4), the FGF-receptor family (FGF-Rs 1 through 4), the Met family (Met, Ron), etc. There are 11 known families of non-receptor type tyrosine kinases including the Src family (Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, Yrk), Abl family (Abl, Arg), Zap 70 family (Zap 70, Syk) and Jak family (Jak 1, Jak 2, Tyk 2, Jak 3). Many of these tyrosine kinases have been found to be involved in cellular signaling pathways leading to pathogenic conditions such as cancer, psoriasis, hyperimmune response, etc. Other roles for tyrosine kinases include cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiating developmental processes in both vertebrates and invertebrates.
Among various types of tumors, e.g., sarcoma, neuroblastoma, breast carcinoma among many others, c-Src has been found to be activated, particularly in colon cancers, especially in those metastatic to the liver (Rosen, N. et al. Analysis of pp60c-Src protein kinase activity in human tumor cell lines and tissues. J Biol Chem 261, 13754-13759 (1986); Bolen, J., Veillette, A., Schwartz, A., DeSeau, V. and Rosen, N. Activation of pp60c-Src protein kinase activity in human colon carcinoma. Proc. Natl Acad. Sci. USA 84, 2251-2255 (1987); Cartwright, C., Kamps, M., Meisler, A., Pipas, J. and Eckhart, W. pp60c-Src activation in human colon carcinoma. J. Clin. Invest. 83, 2025-2033 (1989); Talamonti, M. A., Roh, M. S., Curley, S. A. and Gallick, G. E. Increase in activity and level of pp60c-Src in progressive stages of human colorectal cancer. J. Clin. Invest. 91, 53-60 (1991); Cartwright, C., Coad, C. and Egbert, B. Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis. J. Clin. Invest. 93, 509-515 (1994); Termnuhlen, P. M., Curley, S. A., Talamonti, M. S., Saboorian, M. H. and Gallick, G. E. Site-specific differences in pp60c-Src activity in human colorectal metastases. J. Surg. Res. 54, 293-298 (1993); Mao, W. et al. Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential. Oncogene 15, 3083-3090 (1997)).
Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity can be downregulated by phosphorylation of an amino acid tyrosine at position 530 (Tyr 530 in human c-Src, which is equivalent to Tyr 527 in chicken Src) of the C-terminal regulatory region (Cooper, J., Gould, K., Cartwright, C. and Hunter, T. Tyr 527 is phosphorylated in pp60c-Src: implications for regulation. Science 231, 1431-1434 (1986); Cartwright, C., Eckhart, W., Simon, S. and Kaplan, P. Cell transformation by pp60c-Src mutated in the carboxy-terminal regulatory domain. Cell 49, 83-91 (1987); Kmiecik, T. and Shalloway, D. Activation and suppression of pp60c-Src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 49, 65-73 (1987); Piwnica-Worms, H., Saunders, K. B., Roberts, T. M., Smith, A. E. and Cheng, S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-Src. Cell 49, 75-82 (1987); Reynolds, A. B. et al. Activation of the oncogenic potential of the avian cellular Src protein by specific structural alteration of the carboxy terminus. Embo J. 6, 2359-2364 (1987); Jove, R., Hanafusa, T., Hamaguchi, M. and Hanafusa, H. In vivo phosphorylation states and kinase activities of transforming p60c-Src mutants. Oncogene Res. 5, 49-60 (1989); Bjorge, J. et al. Characterization of two activated mutants of human pp60c-Src that escape c-Src kinase regulation by distinct mechanisms. J. Biol. Chem. 270, 24222-24228 (1995)). It is possible that other mutations and phosphorylation processes involving tyrosine and other amino acids encoded by Src oncogene might be linked to tumorigenesis. For example, in chickens a single point mutation at residues Thr 338, Glu 378, Ile 441 or Arg 95 appears to activate the transforming ability of pp60c-Src (Wang P, Fromowitz F, Koslow M, Hagag N, Johnson B, Viola M. c-Src structure in human cancers with elevated pp60c-Src activity. Br J Cancer Sep; 64(3):531-3, 1991). However, according to the current state of the art, nothing has been identified in the human species that is as important as phosphorylation of Tyr 530 residue. For example, phosphorylation of Tyr 419 is not essential for tumor transformation (Snyder, M. A., Bishop, J. M., Colby, W. W. and Levinson, A. D. Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-Src. Cell 32, 891-901 (1983)). While this Tyr 530 mutation might be responsible for tumor formation it may not be the only cause and there is thus a continuing need to identify and further characterize the c-Src gene and pp60 as targets for drug discovery. The present inventors have surprisingly discovered for the first time that a novel mutation at SRC 531 is responsible for malignant transformation and metastasis. The existence of a mutant form of c-Src (SEQ ID NO. 3) is disclosed that plays a role in Src activation in cancer.
The present invention relates to mutated c-Src, in particular to Src polynucleotides and c-Src polypeptides and methods of using them in fields of diagnosis, therapy, and prevention arts. More specifically, the present invention provides a recombinant nucleic acid or oligonucleotide consisting essentially of SEQ ID NO. 3 and a polypeptide encoded by this nucleic acid (SEQ ID NO. 4). The oligonucleotide having a sequence complementary to the SEQ ID NO. 3 is also provided. Preferably the c-Src oncogene of the invention is truncated and preferably this truncation occurs at the 3xe2x80x2 end. As a result of the truncation the expression of truncated c-Src preferably results in loss of one or more amino acids in the C-terminal end of phosphoprotein pp60c-Src. An isolated DNA molecule is contemplated which comprises a nucleic acid sequence encoding a mutated protein comprising Src protein tyrosine kinase activity, lacking the carboxy-terminal end. Also contemplated is an isolated nucleic acid consisting of the nucleotide sequence of SEQ ID NO. 3 or a contiguous fragment thereof wherein said isolated nucleic acid encodes a polypeptide having the biological activity of tyrosine kinase protein. Also contemplated is an isolated nucleic acid consisting of a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO. 3.
The instant invention also provides a polypeptide of about 400 to 530 amino acids in length and having at least 80% amino acid homology to the mutated c-Src 531 polypeptide of SEQ ID NO. 4, wherein said homologous polypeptide displays tyrosine kinase activity. Accordingly, methods are provided for producing and purifying these polypeptides. These methods include the steps of culturing the c-Src mutant transformed host cell under conditions suitable for the expression of the polypeptide and recovering the mutant c-Src polypeptide from the host cell or the host cell culture.
This invention also provides a method of screening agonist and antagonist compounds for the treatment of mutant Src associated or caused diseases. A method of treating a cancer is provided by administering to cancerous cells exhibiting a c-Src mutation at SRC 531 an effective amount of a compound capable of inhibiting the excess kinase activity resulting from the c-Src mutation or capable of inhibiting expression of the c-Src mutant gene (SEQ ID NO. 3). Preferred compounds of the invention comprise an antisense oligonucleotide, or a preparation of antibodies, or other molecules which specifically bind to c-Src SRC 531 mutant (SEQ ID NO. 3).
Another preferred embodiment of the invention comprises an expression construct for expressing all or a portion of c-Src SRC 531 mutant (SEQ ID NO. 3). Such a construct comprises a promoter; and an oligonucleotide segment having at least one mutated nucleic acid residue of c-Src mutant and located downstream from the promoter, wherein transcription of the segment is initiated at the promoter. A replicable vector comprising the nucleic acid of mutant c-Src is also provided.
The present invention entails a host cell containing a replicable vector or a recombinant host cell having at least one nucleic acid sequence encoding for SRC 531 (SEQ ID NO. 4) mutant as well as a cell line transformed with SRC 531 mutant Src-oncogene (SEQ ID NO. 3). Also contemplated is a host cell comprising the isolated purified nucleic acid corresponding to SRC 531 mutant Src-oncogene.
Various methods are provided for detecting the presence of SRC 531 mutation in Src oncogene contained in a sample. Such methods can include contacting the sample with two primers that are upstream and downstream of SRC 531 region, amplifying the SRC 531 region according to standard procedures, and detecting whether the amplified sequence is present or absent in the nucleic acid sample. Accordingly primers capable of recognizing and binding to SRC 531 region and nucleic acid probes having an affinity to SRC 531 mutated region of Src oncogene are preferred means of supporting such methods. Without limiting to these diagnostic methods a method is provided for detecting SRC 531 mutation whereby a restriction enzyme Sca I is used to recognize the lack or presence of restriction site at the mutated codon. Thus, also envisioned in the present invention is a diagnostic kit for detecting mutant Src oncogene related malignancy in an animal. Such a kit preferably comprises multiple containers wherein included is a set of primers useful for PCR detection of the mutated region of Src oncogene, and optionally a positive control comprising mutated Src sequence and a negative control comprising a non-mutated Src sequence.
The present invention also comprises a transgenic animal such as a mouse whose somatic and germ cells contain a gene (SEQ ID NO. 3) encoding for SRC 531 (SEQ ID NO. 4), said gene operably linked to a promoter, wherein expression of said SRC 531 gene results in the formation of inborn abnormalities or tumors in the mouse.
Preferably, a composition comprising the c-Src mutant polypeptide (SEQ ID NO. 4) is provided in combination with an immune adjuvant. This composition serves as a cancer vaccine comprising as an immunogen at least one immunogenic epitope of the SRC 531 mutant protein.